Myriad catabolic pathways have evolved through time to defend all life forms against harmful exogenous chemicals (xenobiotics) and their metabolites, and many epoxides comprise a particularly sinister group of xenobiotic metabolites. For example, many aromatic and olefinic hydrocarbons are readily oxidized by cytochrome P450 monooxygenases in vivo to form epoxides capable of alkylating nucleic acids. The first line of chemical defense against such epoxides is the liver enzyme epoxide hydrolase. This enzyme hydrolyzes epoxides to form the corresponding vicinal 1,2-diols, which are typically less reactive, less mutagenic, and more rapidly excreted due to increased solubility. Therefore, epoxide hydrolase is the first line of chemical defense against harmful compounds that can damage the genetic material. Strikingly, the cytosolic or soluble isoform of epoxide hydrolase, designated sEH, also plays a greater role beyond that of xenobiotic catabolism and catalyzes the hydrolysis of endogenous fatty acid epoxides such as those derived from arachidonic acid and linoleic acid. The diol products resulting from hydrolysis of these fatty acid epoxides are implicated in pregnancy-induced hypertension and acute respiratory distress syndrome, respectively. Thus, sEH is a potential target for drug design. The structural and chemical basis of epoxide activation in biology is little understood, and our recently-determined X-ray crystal structure of murine sEH provides the first step toward a greater understanding of the epoxide activation and hydrolysis reaction. We propose to build upon the foundation established with the sEH structure. Specifically, we aim: (1) to probe the structure-based mechanism proposed for sEH by determining structures of site-specific sEH variants and enzyme-inhibitor complexes; (2) to determine the crystal structure of human sEH; (3) to explore structure-mechanism relationships for the N-terminal domain of sEH, which unexpectedly exhibits a metal-dependent phosphatase activity. The structural chemistry and biology of this domain will help us explore the evolutionary imperative for the covalent linkage of the activities of epoxide hydrolysis and phosphate hydrolysis within the same macromolecular catalyst.